Analysis of extracellular vesicle DNA at the single‐vesicle level by nano‐flow cytometry

Abstract It has been demonstrated recently that extracellular vesicles (EVs) carry DNA; however, many fundamental features of DNA in EVs (EV‐DNA) remain elusive. In this study, a laboratory‐built nano‐flow cytometer (nFCM) that can detect single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp upon SYTO 16 staining was used to study EV‐DNA at the single‐vesicle level. Through simultaneous side‐scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non‐vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV‐DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA+) EVs varies from 30% to 80% depending on the cell type; (3) external EV‐DNA is mainly localized on relatively small size EVs (e.g. <100 nm for HCT‐15 cell line) and the secretion of external DNA+ EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV‐DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80–200 nm for HCT‐15 cell line); (5) double‐stranded DNA (dsDNA) is the predominant form of both the external and internal EV‐DNA; (6) histones (H3) are not found in EVs, and EV‐DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA+ EVs, and the number of both external DNA+ EVs and internal DNA+ EVs as well as the DNA content in single EVs increase significantly. This study provides direct and conclusive experimental evidence for an in‐depth understanding of how DNA is associated with EVs.

. (a) SSC distribution histogram of a mixture of monodisperse SiNPs of five different diameters ranging between 47 and 123 nm, and fit to a sum of Gaussian peaks.
(b) Measured (black symbol) and Mie theory calculated (black line) SSC intensity versus diameter for SiNPs along with the predicted (red symbol) and Mie theory calculated (red line) SSC intensity versus diameter for EVs. Mie calculation are in excellent agreement with the data. (c) Plot of the calibration curve between the refractive index correctedintensity of Gaussian-fitted SiNPs SSC signal and the EV particle size.
Note: Simulation of side-scattered light detected by the nFCM system was performed with MiePlot v4.6 (a computer program for scattering of light from a single sphere using Mie theory and the Debye series) and Origin 8.6 software (Anal. Chem. 2018, 90, 12768−12775). The maximum half angle (αmax) of the cone of light that can enter the objective lens is defined as NA = nmsinαmax, where NA is the numerical aperture of the objective and nm is the refractive index (RI) of surrounding medium. In the present study, the NA of objective lens was 0.55 and nm was 1.332. Therefore, the calculated αmax is 24°. The sheath flow was ultrapure water, and 1.332 was used as the refractive index of the medium. Considering the refractive index difference between SiNPs (1.463) and EVs (1.400) at 488 nm excitation, the intensity ratio between light scattered by a SiNP to that of an EV of the same particle size was calculated based on the Mie theory for every size of the SiNP standard. Then the predicted SSC intensity for an EV of the same particle size of SiNP was obtained by dividing the SSC intensity of the SiNP by the intensity ratio.
The calibration curve of SSC intensity of EVs versus diameter was constructed based on the measured SiNPs data and upon refractive index correction (c). concentration of 5 μg/mL each was incubated with 6 M SYTO dye or 1 PicoGreen or SYBR Green I for 20 min at 37C. Fluorescence emission spectra were measured on a Spectrofluorometer (F-5301Pc, Shimadzu) with excitation at 488 nm for SYTO 9, SYTO 13, SYTO 16, PicoGreen, and SYBR Green I and with excitation at 532 nm for SYTO 82.   fluorescence versus SSC intensity for an EV preparation before (i) and after 1% Triton X-100 treatment (ii). (b) Bar graphs of the total events, DNA + events, and DNAevents detected in 1 min for EV preparations before and after Triton X-100 treatment. Data are represented as mean ± s.d. (n = 3). Note that SYTO16 staining was carried out after detergent treatment, and the events without detectable SSC signal were ignored.
Note: The threshold levels for both the peak height (a digital discriminator level set to 3 times the standard deviation of the background) and the peak width of 0.2 ms and 0.3 ms were set as the criteria for burst (or peak) identification of SSC and FL signal, respectively.
For each burst that satisfied the criteria, the integrated number of photons (background subtracted) was stored as the burst area for the histogram or dot-plot construction.
According to the criteria, the FL burst area for each identified fluorescent event is calculated by integrating the number of photons detected in its duration (peak width), i.e. ≥ 0.3 ms (3 bins). For those events that only have SSC signal without FL, their fluorescence burst area was calculated by integrating the number of photons detected within 0.2 ms (2 bins) from the starting point of the SSC burst. Therefore, there exist a distinct discontinuity between the "positive" (green-colored events) and "negative" (black colored events) subsets.        Note: The qEV single column (Izon, SP2101516) was equilibrated with 3.5 mL PBS before using. Then 150 μL EV isolate was pipetted onto the column, and fractions were immediately collected with a volume of 200 μL into each tube. PBS was used to elute EVs during the purification process. Based on the manufacturer's instructions, the first five fractions were discarded and the 6th-8th fractions were collected. The 6th-8th fraction and their equal volume mixture were analyzed on the nFCM before and after DNase I treatment.
We can see from the results shown below although there exists some variation in the population ratios of cell-free DNA, debris or DNA -EVs, and DNA + EVs, none of the 6th -8th fraction can provide EV preparation that does not have cell-free DNA. Note: Freshly prepared CCCM from HCT-15 cells (60 mL) was divided equally into five tubes and centrifuged at 100,000 × g for 2 h at 4°C (Optima XE-90 ultracentrifuge with a SW 41Ti rotor, Beckman Coulter). All EV pellets were combined into a centrifuge tube and suspended in 12 mL of PBS, followed by a second ultracentrifugation at 100,000 × g for 2 h at 4°C. Afterwards, the supernatant was discarded, and the EVs were resuspended in 1 mL of PBS. A total of 1 mL EV sample was mixed with 3 mL of 60% iodixanol (OptiPrepTM, 00119) and laid at the bottom of the centrifuge tube, and 1 mL layers of 35%, 30%, 28%, 26%, 24%, 22%, 22%, and 20% iodixanol were subsequently overlaid forming a discontinuous gradient. The sample was ultracentrifuged at 180,000 × g (Optima XE-90 ultracentrifuge with a SW 41Ti rotor, Beckman Coulter) for 16 h. Fractions of 1 mL were collected from the top to the bottom, and the density of each iodixanol fraction was measured by weighting. Next, the sample of different fractions was transferred to a new tube, diluted, washed with PBS (up to 12 mL), and ultracentrifuged at 100,000 × g for 2 h (Optima XE-90 ultracentrifuge with a SW 41Ti rotor, Beckman Coulter). Afterwards, the supernatant was discarded, and the EVs were resuspended in 200 μL of PBS. These samples were stained with SYTO 16 and analyzed on the nFCM before and after DNase I treatment. The particle concentration of each fraction was also measured. We can see that although density gradient ultracentrifugation can efficiently separate cell-free DNA from EVs, the EV-DNA adhered to the outer membrane of EVs was also detached from the surface owing to the harsh separation process. Figure S15. The functionality and specificity of dsDNase and S1 nuclease were verified by using purified Lambda dsDNA and ssDNA oligonucleotides as the substrates.  Note: About 1 × 10 6 HCT-15 cells were collected and centrifuged at 800  g for 5 min at 4°C. Cells were resuspended in 100 μL 4% PFA and incubated at room temperature for 30 min. The PFA was washed away with 1 mL PBS by ultracentrifugation at 800 g for 5 min at 4 °C. Subsequently, cells were resuspended in 100 μL of 0.2% Tween 20, incubated for 15 min at room temperature. Cells were centrifuged at 800  g for 5 min at 4°C, and resuspended in 100 μL PBS. Then, 5 μg/mL of AF488-conjugated mouse anti-human histone H3 antibody (Thermo Fisher, MA531759) was added. The mixture was incubated at 37°C for 30 min and then washed twice with 1 mL PBS by ultracentrifugation at 800  g for 5 min at 4°C. The pellet was resuspended in 500 μL PBS for BD FACSAria IIIu Cytometer analysis.